This parent grant studies an important streptococcal adhesin Fap1 of Streptococcus parasanguinis we discovered few years ago. Interestingly Fap1 is glycosylated by a conserved gene cluster that is also present in pathogenic streptococci and staphylococci. Two specific aims were proposed to characterize this new glycosylation and secretion pathway in the parent grant. Aim 1 was to determine the first step of the Fap1 glycosylation and Aim 2 was to characterize accessory secretion protein SecY2-mediated Fap1 glycosylation step. During the study, we have discovered that Fap1 is a potent antigen and mediates proinflammatory responses via innate immune Toll-Like Receptor 2. TLRs and related innate immune signaling play important roles in pathogenesis of human diseases. In this revision, we will test our hypothesis that Fap1 and Fap1 glycosylation play an important role in S. parasanguinis-mediated TLR signaling and in the pathogenesis of infective endocarditis (IE) as oral streptococci have been strongly associated with the development of IE. We will test our hypothesis by pursuing three sub-aims: Part. A. Determine the function of the Fap1 glycosylation in S. parasanguinis-mediated TLR signaling. Fap1 mediates innate immune response. As Fap1 is a glycoprotein, we will dissect functional domains of Fap1 (glycans and peptides) that are important in TLR signaling. In addition, we will determine the functional contribution of innate immune signaling to Fap1-mediated inflammatory responses. Part. B. Determine the function of Fap1 and Fap1 glycosylation in the pathogenesis of IE. We will evaluate the role of Fap1 and Fap1 glycosylation in the pathogenesis of IE using Fap1 and Fap1 glycosylation deficient mutants constructed in the parent grant in a well-characterized rabbit model of IE. Part. C. Develop a mouse model of IE and determine the functional contribution of innate immunity to the pathogenesis of IE. As genetically modified animals are available for mice, we will establish a mouse model of IE to define the role of TLR2 and its receptor MyD88 in regulating the disease development using TLR2-/- and MyD88-/- mice and their control littermates. Fap1-like proteins are highly conserved in many streptococci and pathogenic straphylcocci and have been implicated in the pathogenesis of IE. Glycosylation and biogenesis of Fap1 is a previously unexplored biosynthetic pathway, therefore establishing the function of Fap1 and its relationship with innate immune system in the disease pathogenesis will provide a unique opportunity to design and develop new therapeutics. Our proposed competitive revision expands the scientific scope of the parent grant. Supporting of the proposal will allow us to hire additional staff and contract additional needed skills to stimulate economy, the objectives of the Recovery Act. PUBLIC HEALTH RELEVANCE: Our proposed competitive revision fits nicely with the scope of our parent grant and will expand our study on bacterial pathogenesis and identify new targets for the design of therapeutics. In addition, supporting of the proposal will allow us to hire additional staff (a graduate student and a Research Associate), thus, our revision will not only accelerate the tempo of scientific research but also allow for job creation, the objectives of the Recovery Act.